New Asian types of Varroa destructor: a potential new ...
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Apidologie 41 (2010) 181–193 Available online at:c© INRA/DIB-AGIB/EDP Sciences, 2009 www.apidologie.orgDOI: 10.1051/apido/2009068
Original article
New Asian types of Varroa destructor: a potential new threatfor world apiculture*
Maria Navajas1, Denis L. Anderson2, Lilia I. de Guzman3, Zachary Y. Huang4,Jeremy Clement1, Ting Zhou5, Yves Le Conte6
1 INRA, UMR CBGP (INRA/IRD/Cirad/Montpellier SupAgro), Campus International de Baillarguet, CS 30016,34988 Montferrier-sur-Lez Cedex, France
2 CSIRO Entomology, PO Box 1700, Canberra, ACT 2601, Australia3 USDA/ARS, Honey-Bee Breeding, Genetics and Physiology Laboratory, 1157 Ben Hur Road, Baton Rouge,
LA 70820, USA4 Department of Entomology, Michigan State University, East Lansing, MI 48824, USA
5 Institute of Apicultural Research, Chinese Academy of Agricultural Sciences, Beijing 100093, China6 INRA, UMR INRA/UAPV Abeilles et Environnement, Laboratoire Biologie et Protection de l’Abeille,
Site Agroparc, Domaine Saint-Paul, 84914 Avignon Cedex 9, France
Received 22 January 2009 – Revised 17 July 2009 – Accepted 21 August 2009
Abstract – The invasion of the Western honey bee, Apis mellifera, by Varroa destructor is attributed totwo mitochondrial haplotypes (K and J) that shifted last century from their primary host the Eastern honeybee, A. cerana, in north-east Asia. Here, mitochondrial DNA sequences (cox1, cox3, atp6 and cytb: 2700base pairs) were obtained from mites infesting both Eastern and Western honeybees (respectively 21 and 11colonies) from Asia including regions where the shifts first occurred. A total of eighteen haplotypes wereuncovered in Asia (11 on A. cerana and 7 on A. mellifera). Two new variants of the K haplotype and two ofthe J haplotype were found on Western honeybees in what appeared to be well-established infestations. Newhaplotypes may represent a potential threat to A. mellifera worldwide. The extreme lack of polymorphism inthe K and J haplotypes outside of Asia, can now be plausibly explained as being due to genetic ‘bottlenecks’that occurred in Asia before and after mites shifted from their original Eastern honeybee host.
Apis mellifera / Apis cerana / Varroa / mitochondrial DNA/diversity
1. INTRODUCTION
The varroa mite, Varroa destructor (Acari:Varroidae), is a well-adapted parasite of theEastern honeybee (Apis cerana) in northernregions of mainland Asia. It also dramati-cally expanded its distribution when it shiftedhost to the Western honeybee (A. mellifera)in the last century. Prior to and long after theshift, V. destructor was mistaken for V. ja-cobsoni, a morphologically similar speciesfirst described from A. cerana in Indonesia(Oudemans, 1904). It was not until 2000, fol-
Corresponding author: M. Navajas,[email protected]* Manuscript editor: Klaus Hartfelder
lowing a comprehensive molecular and mor-phological studies of so-called V. jacobsonimites on A. cerana throughout Asia, that it wasrecognized as a distinct species (Anderson andTrueman, 2000). In that particular study, 18different mite haplotypes [mites with distinctmitochondrial (mt) DNA sequences] were dis-covered, nine of which were members of V. ja-cobsoni, six of V. destructor, and the iden-tities of three others were unresolved. Eachhaplotype was named after the country or is-land in which it was first found, e.g., the China1 (C1) haplotype was found in China. Onemore haplotype of V. destructor has subse-quently been found on A. cerana in China
Article published by EDP Sciences
182 M. Navajas et al.
using the same marker (China 2 or C2) (Zhouet al., 2004).
Of the eight known haplotypes of V. de-structor on A. cerana, only Japan 1 (J1) andKorea 1 (K1) have successfully colonizedA. mellifera (Anderson and Trueman, 2000;Solignac et al., 2005). J1 first shifted from A.cerana to A. mellifera in Japan last century fol-lowing the introduction of A. mellifera (Sakaiand Okada, 1973). It then spread on A. mel-lifera into Thailand and Brazil (Anderson andTrueman, 2000) and then into North America(de Guzman et al., 1999). According to deGuzman et al. (1997) and Oldroyd (1999), K1first switched from A. cerana to A. melliferanear Vladivostok (north of the Korean Penin-sula) following the introduction of A. mellif-era from Ukraine during the late 1950s (Crane,1978). It thereafter spread on A. melliferainto Europe in the 1960s (Crane, 1978), thenaround the world, arriving in North America in1987 (de Guzman et al., 1997; Oldroyd, 1999;Sammataro et al., 2000). On A. mellifera, J1and K1 are partially isolated clones (Solignacet al., 2005) for reasons that are not under-stood.
Here, we sought to gain further insights intothe invasion of A. mellifera by V. destructor byidentifying and genotyping isolates of V. de-structor infesting both A. cerana and A. mellif-era in regions where the J1 and K1 host shiftsfirst occurred as well as in a broader area of V.destructor’s natural geographic range in Asia.The aim of our study was to obtain insightsinto the following questions: (i) how variableis V. destructor on its primary host (A. cerana)and its new host (A. mellifera) in Asia wherethe J1 and K1 host shifts occurred? and (ii) areJ1 and K1 on A. mellifera derived from differ-ent mite populations, or do they simply reflecthaplotypes randomly sampled in a single largeVarroa population?
2. MATERIALS AND METHODS
Female Varroa mites were collected from 21 A.cerana and 11 A. mellifera colonies in several Asiancountries where V. destructor is known to occuralone (China, Japan, Korea, far East Russia, Taiwanand North Vietnam) or sympatric with V. jacobsoni(Thailand) (Tab. I). Several mites per colony were
collected alive from the bee brood and preserved in95% ethanol until needed for DNA analysis. In mostcases, a single bee colony was sampled at one lo-cality, the exceptions being where A. mellifera andA. cerana colonies were hived next to each otherin the same apiary, as in Nanchang (China), where8 colonies were sampled (5 A. cerana and 3 A. mel-lifera), or where V. destructor and V. jacobsoni ex-ist sympatrically as in Chang Mai, Thailand where5 colonies were sampled. In all cases, three miteswere molecularly analyzed from each colony sam-pled, except in the case where 10 mites were ana-lyzed from a single A. mellifera colony from ChangMai (Thailand).
Total DNA was extracted from single mites us-ing the DNeasy tissue Kit (Qiagen, USA), follow-ing manufacturer’s instructions as indicated for thespin-column protocol from Cultured Animal Cellswith the following slight modifications: (i) all vol-umes were reduced to half; (ii) incubation was at70 ◦C for 3 h; (iii) final elution was in 50 μL ofDNase-free water. Four fragments from either thecox1, cytochrome oxidase III (cox3), ATP synthase6 (atp6) and cytochrome b (cytb) mitochondrialgenes were amplified by the polymerase chain re-action (PCR) from each V. destructor mite whereasonly fragments of the cox1, cox3, atp6 genes (samesequences as above), were amplified from V. jacob-soni mites (Tab. I). The PCR primers (Tab. II) weredeveloped from published sequence of the completemtDNA genome of V. destructor (GenBank Ac-cession No. AJ493124.1; Evans and Lopez, 2002;Navajas et al., 2002). A 458 base pair (bp) fragmentof the cox1 gene was also amplified for each miteusing C1/C1R primers defined on sequences pub-lished by (Anderson and Fuchs, 1998). Reactionswere carried out in 25 μL of PCR solution con-taining 2.5 μL of 10X buffer, 1U Taq polymerase(Qiagen), 0.25 mM of each dNTP, 0.5 μM of eacholigonucleotide primer, 2.5 mM of MgCl2 and 2 μLof template DNA. Samples were denatured at 94 ◦Cfor 4 min and then PCR was done for 35 cyclesof 30 s denaturation at 94 ◦C, 30 s annealing (seeTab. II for annealing temperature for each fragment)and 1 min extension at 72 ◦C. PCR products werecleaned-up using the ExoSAP-IT� reagent and sub-sequently directly sequenced in two directions us-ing the BigDye� Terminator method (Parkin Elmer,Foster City, CA, USA) in an ABI PRISM 377 au-tomated DNA sequencer (Applied Biosystems Inc).Sequences obtained using the C1/C1R primers wereused to assign mites to a particular cox1 haplo-type for direct comparison with haplotypes found
Varroa destructor diversity in Asia 183Ta
ble
I.O
rigi
n,co
llec
tion
deta
ils
and
iden
tity
ofVa
rroa
dest
ruct
or(V
D)
and
V.ja
cobs
oni
(VJ)
isol
ates
exam
ined
from
Api
sce
rana
(Ac)
and
Api
sm
elli
fera
(Am
)(s
hade
d)co
llec
ted
inA
sia.
Mit
esw
ere
assi
gned
toa
know
n(e
.g.V
1)or
new
hapl
ogro
ups
base
don
part
ial
nucl
eoti
dese
quen
ce(4
58bp
)of
thei
rm
tDN
Aco
x1ge
ne(A
nder
son
and
Tru
eman
,200
0).H
aplo
type
sw
ere
also
furt
her
defi
ned
(e.g
.V1-
2)ba
sed
onco
ncat
enat
ednu
cleo
tide
sequ
ence
sof
frag
men
tsof
thei
rm
tDN
Aco
x1,c
ox3,
atp6
and
cytb
gene
sequ
ence
s(2
700
bp)
obta
ined
inth
isst
udy.
Mite
Bee
Col
lect
ion
Col
onie
sM
ite M
tDN
A c
ox1
Con
cate
nate
d
Cou
ntry
Loc
ality
spec
ies
host
year
Col
lect
orex
amin
edH
a plo
type
(1)
mtD
NA
ha p
loty
p eco
x1at
p 6co
x3cy
tb
Chi
naK
unm
ing,
Yun
nan
Prov
.V
DA
c20
02D
. L. A
nder
son
C2
C2-
1G
Q37
9067
GQ
3791
24G
Q37
9086
GQ
3791
05
Chi
naD
ayao
Co.
Yun
nan
Prov
.V
DA
c20
02Z
. Hua
n g1
C3
(new
)C
3-1
GQ
3790
68G
Q37
9125
GQ
3790
87G
Q37
9106
Chi
naX
ishu
anba
nna,
Yun
nan
Prov
.V
DA
m20
02Z
. Hua
ng1
K1
K1-
4G
Q37
9060
GQ
3791
17G
Q37
9079
GQ
3790
98
Chi
naX
ishu
anba
nna,
Yun
nan
Prov
.V
DA
c20
02Z
. Hua
ng1
V1
V1-
2G
Q37
9062
GQ
3791
19G
Q37
9081
GQ
3791
00
Chi
naZ
huha
i, G
uang
dong
Pro
v.V
DA
c20
01Z
. Hua
ng1
C1
C1-
1G
Q37
9065
GQ
3791
22G
Q37
9084
GQ
3791
03C
hina
Zho
ngsh
an, G
uang
dong
V
DA
c20
02Z
. Hua
ng1
C1
C1-
2G
Q37
9066
GQ
3791
23G
Q37
9085
GQ
3791
04
Chi
naN
anch
ang,
Jia
ngxi
Pro
v.V
DA
c20
04Y
. Le
Con
te5
K1
K1-
3*
**
*
Chi
naN
anch
ang,
Jia
ngxi
Pro
v.V
DA
m20
04Y
. Le
Con
teK
1-2
GQ
3790
57G
Q37
9114
GQ
3790
76G
Q37
9095
Chi
naH
unan
Pro
v.V
DA
c20
04Y
. Le
Con
te1
K1
K1-
3G
Q37
9059
GQ
3791
16G
Q37
9078
GQ
3790
97Ja
pan
Tok
yo (
Tam
agaw
a U
ni)
VD
Ac
1994
D. L
. And
erso
nJ1
J1-2
GQ
3790
70G
Q37
9127
GQ
3790
89G
Q37
9108
DV
o yk oT
na pa JA
m19
96T
. Yos
hida
J1J1
-6G
Q37
9074
GQ
3791
31G
Q37
9093
GQ
3791
12
Japa
nT
okyo
(3)
VD
Am
2000
T. G
otoh
1K
1K
1-1
**
**
Jap a
nM
achi
daV
DA
c19
98A
. Syl
vest
er1
J1J1
-3G
Q37
9071
GQ
3791
28G
Q37
9090
GQ
3791
09Ja
pan
Shik
oku
VD
Ac
1996
T. Y
oshi
da1
J1J1
-4G
Q37
9072
GQ
3791
29G
Q37
9091
GQ
3791
10D
VluoeS
aeroK
Am
1996
T. S
telz
er1
K1
K1-
1G
Q37
9056
GQ
3791
13G
Q37
9075
GQ
3790
94R
ussi
aV
ladi
vost
okV
DA
m19
95D
. L. A
nder
son
1K
1K
1-1
**
**
Tai
wan
Tai
chun
g(3)
VD
Am
2002
Chy
i-C
hen
Ho
1J1
J1-1
GQ
3790
69G
Q37
9126
GQ
3790
88G
Q37
9107
Tha
iland
Ban
g C
hang
tay
VD
Ac
2003
M. N
avaj
as1
V1
V1-
4G
Q37
9064
GQ
3791
21G
Q37
9083
GQ
3791
02T
haila
ndB
ang
Cha
ngta
yV
J(2)
Ac
2003
M. N
avaj
as1
L1
L1-
1G
Q38
7678
GQ
3876
76G
Q38
7674
-
Tha
iland
Chi
ang
Mai
VJ(2
)A
c20
03M
. Nav
a jas
2L
1L
1-2
GQ
3876
79G
Q38
7677
GQ
3876
75-
Tha
ïland
C
hian
g M
aiV
DA
m19
97L
. de
Guz
man
1J1
J1-5
GQ
3790
73G
Q37
9130
GQ
3790
92G
Q37
9111
Tha
ïland
C
hian
g M
ai
VD
Ac
2003
M. N
ava j
as2
V1
V1-
3G
Q37
9063
GQ
3791
20G
Q37
9082
GQ
3791
01D
Viona
Hmantei
VA
c19
96L
. de
Guz
man
1V
1V
1-1
GQ
3790
61G
Q37
9118
GQ
3790
80G
Q37
9099
DV
io naH
man te i
VA
m19
96L
. de
Guz
man
1K
1K
1-2
GQ
3790
58G
Q37
9115
GQ
3790
77G
Q37
9096
Gen
Ban
k ac
cess
ion
num
bers
1 H
aplo
type
nam
es h
ave
been
abb
revi
ated
: Chi
na 1
(C
1), C
hina
2 (
C2)
, Chi
na 3
(C
3), J
apan
1(J
1), V
ietn
am 1
(V
1), K
orea
1 (
K1)
, Lao
s 1
(L1)
.
* Se
quen
ces
iden
tical
as
othe
r sa
mpl
es h
avin
g th
e sa
me
hapl
otyp
e.
3 Mite
s is
sued
fro
m th
e sa
me
popu
latio
n us
ed in
Sol
igna
c et
al.
(200
5). T
he o
btai
ned
sequ
ence
s ar
e us
ed h
ere
for
hapl
otyp
e co
rres
pond
ence
with
the
prev
ious
K1-
1 ( T
okyo
) an
d J1
-1 (
Tai
chun
g) h
aplo
typ e
s fo
und
on A
. mel
life
ra.
2 H
a plo
typ e
det
erm
ined
on
basi
s of
163
5 bp
fra
gmen
ts o
f th
e m
tDN
A c
ox1
,cox
3an
dat
p6ge
nes.
1 3K
1
1 1
184 M. Navajas et al.
Table II. DNA amplified fragments to study variation in Varroa destructor samples. Amplified gene frag-ment, product size base pairs (bp) and annealing temperatures (Ta) are indicated. PCR primers defined basedon the complete mitochondrial genome sequence of V. destructor (Navajas et al., 2002).
Fragment Primer name Primer sequences (5’-3’) Size (bp) Ta (◦C)
cox1 10KbCOIF1 CTT GTA ATC ATA AGG ATA TTG GAAC 929 51
6,5KbCOIR AAT ACC AGT GGG AAC CGC
atp6-cox3 16KbATP6F GAC ATA TAT CAG TAA CAA TGAG 818 51
16KbCOIIIR GAC TCC AAG TAA TAG TAA AACC
cytb 10KbCytbF-1 GCA GCT TTA GTG GAT TTA CCT AC 985 52
10KbCytbPRIM CTA CAG GAC ACG ATC CCA AG
cox1* C1 GCG GTT CCC ACT GGT ATT 458 52
C1R AAT ACC AGT GGG AAC CGC
*Fragment partially included in the 929 nucleotides long cox1 sequence and used here to sort out samplesaccording to the published haplotypes (Anderson and Trueman, 2000).
in previous studies (Anderson, 2000; Zhou et al.,2004; Solignac et al., 2005), whereas sequences ob-tained using the other primers allowed the detectionof variation within the haplotypes defined using cy-tochrome oxidase I (cox1) sequences (458 bp).
Sequences were assembled using the STADENpackage 1.6 (http://staden.sourceforge.net/) andedited with BioEdit V.5.0.6 software (http://www.mbio.ncsu.edu/BioEdit/bioedit.html) (Hall, 1999).Sequence alignments were initially performed us-ing ClustalW version 1.8 (Thompson et al., 1994)and optimized by visual inspection. Sequences ob-tained from individual mites were concatenated intoa single data set to increase the power of haplo-type relatedness reconstruction. Concatenated se-quences of the cox1, cox3, atp6 and cytb genes ofeach V. destructor haplotype (2700 nucleotides) wasaligned by eye without the need for numerical al-gorithms. Concatenated sequences of fragments ofthe cox1, cox3 and atp6 genes from V. jacobsonihaplotype contained 1635 nucleotides (cytb PCRprimers failed to amplify V. jacobsoni DNA). Identi-cal sequences were grouped and defined as one hap-lotype. Standard sequence diversity indices (Nei,1987) were computed including A (number of al-leles = variable haplotypes), S (number of segre-gating sites = variable nucleotide positions), andπ (nucleotide diversity) using the DnaSP 4.10.4package (http://www.ub.es/dnasp/) (Rozas et al.,2003). Intraspecific haplotype genealogies based onthe 2700 bp V. destructor sequences were recon-structed. Gene genealogies were first inferred bycomputing haplotype network methods using theTCS program v2.1 (Clement et al., 2000), which
essentially gave the same information as a phy-logenetic reconstruction based on the Neighbour-Joining methods as implemented in the MEGAPackage version 3.1 (Kumar et al., 2004). Only theresulting phylogenetic tree is presented here.
3. RESULTS
3.1. Mite variation based on a 458 bpfragment of the mitochondrial cox1gene
Seven different sequences were identifiedamong DNA fragments amplified from a totalof 103 Varroa mites collected from 32 coloniesusing the previously defined C1/C1R primers.Five sequences were identical to either thepublished China 1 (C1), China 2 (C2), Japan 1(J1), Korea 1 (K1) or Vietnam 1 (V1) haplo-types of V. destructor (Anderson and Trueman,2000; Zhou et al., 2004) (GenBank accessionnumbers: AF106900, AY372063, AF106897,AF106899 and AF106901, respectively). Asixth sequence was identical to a Laos 1 (L1)haplotype of V. jacobsoni, which was first de-scribed by Zhou et al. (2004). A new sequencewas also identified and is hereafter referred toas the China 3 (C3) haplotype of V. destruc-tor. Only V. destructor haplotypes were de-tected in geographical regions (China, Japan,Korea, Russia, Taiwan and Northern Vietnam)where V. destructor had been previously found
Varroa destructor diversity in Asia 185
Figure 1. Geographical distribution of Varroa destructor mitochondrial haplotypes on Apis cerana and A.mellifera (in bold). All haplotypes were identified as V. destructor except two L1 haplotypes, which belongto V. jacobsoni (circled) from Thailand. The V. destructor haplotypes found on A. mellifera elsewhere areK1-1 and J1-1 (Solignac et al., 2005). Solid line indicates approximate range of V. destructor and dottedline is that of V. jacobsoni, after Anderson and Trueman (2000) and data provided by D.L. Anderson.
to occur alone (Tab. I, Fig. 1). Both V. destruc-tor and V. jacobsoni haplotypes were foundin Thailand where both mites had been pre-viously found sympatrically (Fig. 1). In allcases, the three mites studied per colony hadidentical mitochondrial sequences as did the10 mites analyzed from an A. mellifera colonynear Chang Mai (Thailand).
3.2. Mite variation based onconcatenated mtDNA sequences
The concatenated sequences revealedgreater genetic variation among mites (18haplotypes; Tab. III) than was revealed by thesequences of the 458 bp cox1 fragment alone(7 haplotypes, see previous section). Hence,mites with identical cox1 sequences were re-garded as members of the same ‘haplogroup’,of which there were 7 and, mites of the samehaplogroup that showed variation within their
concatenated sequences were regarded asvariants of a particular haplogroup.
Among mites collected from A. melliferain regions of northeast Asia where only V.destructor, not V. jacobsoni, has been previ-ously reported, 3 variants were found of theK1 haplogroup (K1-1, K1-2, and K1-4) and2 variants of the J1 haplogroup (J1-1 and J1-6). Among mites collected from A. ceranain this same region, 1 variant was found ofthe K1 haplogroup (K1-3), 3 of the J1 hap-logroup (J1-2, J1-3 and J1-4), 2 of the V1 hap-logroup (V1-1 and V1-2) and 1 variant eachof the C2 and C3 haplogroups (C2-1 and C3-1). Hence, only variants of V. destructor werefound on A. mellifera and A. cerana in theseregions. Also, only variants of the K1 and J1haplogroups were found on A. mellifera andnone of those variants were found on A. cer-ana. Among mites collected from Thailand,where V. destructor and V. jacobsoni coexist,two variants were found of the V1 haplogroupon A. cerana (V1-3 and V1-4) and two of the
186 M. Navajas et al.
Tabl
eII
I.N
ucle
otid
epo
siti
ons
ofal
igne
dm
itoc
hond
rial
sequ
ence
sof
four
gene
s(c
ox1,
atp6
,cox
3an
dcy
tb)
that
dist
ingu
ish
hapl
otyp
esof
Varr
oade
stru
ctor
.D
ots
indi
cate
iden
tical
nucl
eotid
eas
inth
efi
rst
sequ
ence
.F
igur
esco
rres
pond
topo
sitio
nnu
mbe
rsin
the
com
plet
em
itoch
ondr
ial
sequ
ence
ofV.
dest
ruct
or(A
J493
124.
1).M
ite
sam
ples
wer
eco
llec
ted
onei
ther
Api
sm
elli
fera
(sha
ded
hapl
otyp
eco
de)
orA
pis
cera
na.
Hap
loty
peLo
calit
y (c
ount
ry)
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
22
22
24
44
44
44
44
44
44
44
44
44
44
49
99
90
00
00
00
00
00
00
00
00
44
44
46
66
67
77
88
88
90
01
23
01
11
12
23
45
55
55
55
66
77
77
88
99
00
00
11
23
44
44
55
56
72
37
79
35
57
14
80
04
67
35
90
19
46
67
55
10
03
34
45
93
60
16
75
76
92
46
75
97
33
57
93
89
31
24
36
78
69
46
98
39
53
41
59
26
75
03
60
93
07
78
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Varroa destructor diversity in Asia 187
L1 haplogroup (L1-1 and L1-2) also on A. cer-ana. A variant of the J1 haplogroup (J1-5) wasfound on A. mellifera. Hence, only V. destruc-tor was found on A. mellifera in Thailand butboth V. destructor and V. jacobsoni were foundon A. cerana. Again, none of the mite variantsfound on A. cerana was found on A. melliferaand vice versa.
The concatenated sequences of the K1-1and J1-1 variants described are those of V. de-structor collected from Taichung (Taiwan) andTokyo (Japan), respectively (Tab. I), which hadbeen previously identified using microsatel-lites as K1 and J1 types by Solignac et al.(2005).
GenBank accession numbers for the se-quences of the 4 partial mitochondrial genesdescribed here are given in Table I and theirorigin of detection in Asia is shown in Fig-ure 1.
3.3. Nucleotide diversity
Standard sequence diversity indices (π)were computed from the mitochondrialconcatenated sequences (2700 bp) for V. de-structor collected on both A. mellifera andA. cerana (Tab. IV). The indice (π) for the11 V. destructor haplotypes collected fromA. cerana was π = 0.0078, whereas for the 7V. destructor haplotypes carried by A. mellif-era it was π = 0.0047, and for V. destructorisolates collected from both A. cerana andA. mellifera it was π = 0.0075. Sequencesof only 2 individuals of V. jacobsoni wereanalyzed in this study and indices of diversitywere not estimated. The two haplotypes (L1-1and L1-2) differ by 2 point mutations in thecox3 sequence.
3.4. Phylogenetic relationships
Sixty-six (66) nucleotides of the concate-nated V. destructor sequences were polymor-phic. An unrooted neighbour-joining tree ofall V. destructor sequences showed two well-supported clades (Fig. 2), the first of whichgrouped variants of the Korea and Japan hap-logroups and, the second, variants of the China Ta
ble
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188 M. Navajas et al.
Figure 2. Phylogenetic relationships among haplotypes belonging to six different haplogroups (K1, J1, C1,C2, C3, V1) of Varroa destructor collected from two hosts, Apis cerana (Ac) and A. mellifera (Am) in Asia.Each haplotype is preceded by Am or Ac according to the host where the mite was sampled. Haplotypeswere defined by sequences of 2700 nucleotides long of the mitochondrial genome spanning 4 genes (cox1,cox3, atp6 and cytb).
and Vietnam haplogroups. A common lineagegrouped all variants of the V1 haplotype to-gether with the two variants of the C1 hap-lotype (95% bootstraps score), the two otherChina haplotypes (C2 and C3) remaining apartfrom this grouping.
4. DISCUSSION
Our study shows that the original K1 andJ1 haplotypes of V. destructor on A. mellif-era worldwide were derived from two dis-tinct mite populations (represented by our K1and J1 haplogroups, respectively) that infestA. cerana in north-east Asia. Both populationscontain much more genetic variation than pre-viously realised and newly discovered haplo-types in each population have successfully col-onized A. mellifera in Asia, but have not yet
spread from that region. These new haplotypesnow represent new potential threats to A. mel-lifera outside of Asia.
4.1. Sensitivity of mitochondrialmarkers for detecting Varroavariation
Most variation detected to date among var-roa isolates has been found using a singlemtDNA marker. This marker, developed byAnderson and Fuchs (1998), was widely usedby Anderson and Trueman (2000) to clarifythe level of genetic variation among varroamites on A. cerana throughout Asia and to helpresolve varroa taxonomy. It has also been usedin several more recent studies on varroa varia-tion (Koeniger et al., 2002; Zhou et al., 2004;
Varroa destructor diversity in Asia 189
Solignac et al., 2005; Warrit et al., 2006). Themarker detects sequence variation in a 458DNA base-pair region of the mt cox1 gene. Itcan readily resolve an individual mite to thespecies level, as variation in this gene region isless than 2% among mites of the same speciesand more than 6% among mites of differentspecies (Anderson and Trueman, 2000).
Here, we employed new longer mitochon-drial sequences to determine how useful thecox1 marker is for detecting varroa variation. Itwas not surprising that concatenated sequencedata from the new sequences were far moresensitive at detecting variation than the cox1marker alone. However, it became obviousthat, when used alone, the cox1 marker wasable to define particular populations of mites(‘haplogroups’), whose members (haplotypes)were identified using the new markers. Hence,the combined use of the cox1 marker and thenew markers allows for detection of geneticvariation both within and between differentvarroa populations. The sensitivity of the newmt sequences at detecting genetic variationfalls between that of the less sensitive cox1marker and the more sensitive microsatellitesused by Solignac et al. (2005).
4.2. Genetic variation among varroamites on A. cerana in north-eastAsia
We uncovered significant genetic diversityamong varroa mites on A. cerana in north-east Asia (Tab. I; Tab. III). Although only 1new haplogroup (C3 in Tab. I) was detected,10 haplotypes were uncovered within hap-logroups in which no genetic variation waspreviously known. Thus, we showed that var-roa mites are more genetically variable on A.cerana in Asia than previously recognized.
Our results also showed that some closely-related varroa haplotypes on A. cerana hadsimilar and quite restricted geographical dis-tributions. For example, C1-1 and C1-2 inFigure 2 were both collected from the sameprovince of Guangdong in China. On the otherhand, members of some haplogroups such asthose found in Thailand, China and Vietnam,were widely distributed. It is possible that the
artificial movement of A. cerana in Asia by hu-mans may have influenced such wide distribu-tions. Hence, a better way of determining nat-ural distributions may come from examiningthe associations of particular mite haplogroupswith particular genotypes of A. cerana, as pre-vious studies have noted that the biogeographyof varroa haplotypes in Asia seems to correlatewith the biogeography of A. cerana haplotypes(Anderson and Trueman, 2000; Warrit et al.,2006). Interestingly, none of the varroa haplo-types detected here on A. cerana was found onA. mellifera and vice-versa.
4.3. Genetic variation among varroamites on A. mellifera in north-eastAsia
As well as redefining the taxonomy ofvarroa, Anderson and Trueman (2000) alsoshowed that K1 and J1 haplotypes of V. de-structor were the only varroa mites to havesuccessfully invaded and colonized A. mellif-era globally. Solignac et al. (2005) verifiedthose findings and further showed that therewas almost no genetic variation between K1and J1 (our K1-1 and J1-1) on A. mellifera forreasons that were not clear.
Here, V. destructor was the only varroaspecies detected on A. mellifera in north-eastAsia and all haplotypes found were membersof the K1 and J1 haplogroups. However, four(4) new haplotypes not previously reported onA. mellifera were also found (Tab. I; Tab. III).A new K1-2 haplotype was found on A. mel-lifera in Vietnam, a K1-2 and a K1-4 in China,a J1-5 in Thailand, and a J1-6 in Japan. Thepresence of these haplotypes in A. melliferacolonies at these locations is not unequivocalevidence that they have successfully colonizedthe colonies. Only mites that are able to repro-duce on A. mellifera brood could successfullycolonize and we do not have that informationon the new haplotypes. The presence of varroamites in an A. mellifera colony in Asia couldsimply indicate that the mites invaded thecolonies from a local A. cerana colony withoutcolonizing, as has been shown to occur for theV1-1 haplotype of V. destructor in Vietnam,a Java haplotype of V. jacobsoni in Indonesia
190 M. Navajas et al.
and for three unique varroa genotypes in thePhilippines (Anderson, 1994; Fuchs et al.,2000). However, there is convincing evidencefrom our studies that at least 2 of the newhaplotypes (J1-5 and K1-2) have successfullycolonized A. mellifera in Asia. The most con-vincing evidence is the presence of J1-5 in A.mellifera colonies in Thailand. No member ofthe J1 haplogroup has ever been reported onA. cerana outside of Japan and certainly notin Thailand. J1-1 has been reported outsideof Japan on A. mellifera, but it shows almostno genetic variation (Solignac et al., 2005).It has been known that a member of the J1haplogroup has been present on A. mellifera inThailand since 1997 (Anderson and Trueman,2000). This same member was reported thereagain in 2001 (Warrit et al., 2006). Here wehave shown that that member is J1-5, not J1-1. The only way J1-5 could have survived for4 years in Thailand is if it could colonize A.mellifera. In our study we collected 10 mitesfrom several different pupae in a single A. mel-lifera colony in Thailand and each was J1-5, which is way above the expected detectionthreshold if those mites were simply casual in-vaders of the colony (Anderson, 1994). Hence,it is clear that J1-5 has colonized A. melliferain Asia. Our observations corroborate anec-dotal evidence that A. mellifera was importedinto Thailand from Japan via Taiwan as partof a bee extension program (Anderson, unpub-lished data).
The presence of K1-2 on A. mellifera inVietnam and China provides less convincingevidence, but still substantial support, that hap-lotypes other than K1-1 and J1-1 have colo-nized A. mellifera in Asia. The detection ofthis haplotype in A. mellifera colonies wasabove the detection threshold expected for acasual invader and, interesting, like all othervarroa haplotypes that have successfully in-vaded A. mellifera, we failed to detect K1-2on A. cerana.
4.4. Insights into the geographicalorigins and invasive biologyof V. destructor
Although there was no clear geographicalstructure of most mitochondrial haplotypes of
V. destructor in Asia, most haplotypes of theJ1 haplogroup (5 out of the 6) were found inJapan, confirming previous claims that this re-gion was the origin of the J type (de Guzmanet al., 1997; Anderson and Trueman, 2000). Asfor J1-1, evidence indicates it shifted to A. mel-lifera in Japan and was subsequently trans-ported to Brazil during the 1970’s (de Jong andGonçalves, 1981; De Jong et al., 1982).
Whether or not all haplotypes of the K1haplogroup colonizing A. mellifera originatedfrom a discrete geographic region of Korea, assuggested by Warrit et al. (2006), needs fur-ther investigation. Here, we found K1-1 onlyon A. mellifera in Korea (Seoul) and Russia(Vladivostok), which lends support the hy-pothesis that K1-1 first colonized A. melliferain Far-Eastern Russia (Danka et al., 1995) fol-lowing a host-shift somewhere near Korea. Ev-idence suggests that the host-shift was madepossible because mites of a single geneticlinage (K1-1) were able to invade an A. mellif-era colony from a colony of their indigenousA. cerana and were then able to lay eggs andproduce offspring on the A. mellifera brood(Solignac et al., 2005). Our inability to findK1-1 on A. cerana in Asia lends further sup-port to this hypothesis. After shifting to A. mel-lifera, K1-1 was then moved out of Asia intoEurope, from where it spread globally.
By discovering new haplotypes of V. de-structor colonizing A. mellifera in Asia, ourstudies suggest that the colonization of A. mel-lifera in Asia by new haplotypes is depen-dent on the length of exposure of those haplo-types to A. mellifera. Perhaps longer exposureeventually results in one type being selectedfor their ability to reproduce on A. mellifera.Given this scenario, we predict that other typesof V. destructor, including those that are cur-rently known to be non-reproductive on A.mellifera, will eventually colonize A. melliferain Asia. Indeed, it is possible that such typeshave existed before in some parts of Asia, butmay have completely exterminated A. mellif-era and hence themselves.
In contrast to the V. destructor polymor-phism unravelled in Asia, the V. destructorcolonizing A. mellifera worldwide (K1-1 andJ1-1) shows a complete lack of genetic di-versity. These results are consistent with the
Varroa destructor diversity in Asia 191
hypothesis of a bottleneck that occurred at thevery beginning of the invasion of the West-ern honeybee by V. destructor as suggested by(Solignac et al., 2005). This scenario may haveresulted from two independent events, one foreach of the J and K types belonging to two dis-tinct monophyletic haplogroups (Fig. 2). Dueto varroa’s rapid growth rate (about 12-fold in-crease in mite numbers yearly) (Martin, 1998),the situation is hardly compatible with the ero-sion of variability of V. destructor after colo-nizing A. mellifera colonies. A strong foundereffect was also confirmed by the fact that J1-5in Thailand, K1-2 in Vietnam and China, andJ1-6 in Japan, which reproduce on A. mellif-era, have been only found in Asia.
One of the main conclusions that haveemerged from wider studies on invasivespecies is that the successful establishment ofan exotic species into a new range is quite im-probable and that many failed attempts occurbefore a successful invasion develops (Nivak,2007). It has been often hypothesized that aselection of ad hoc genotypes operates at thebeginning of an invasive process (Facon et al.,2006), and it can be speculated that the geno-types having invaded A. mellifera worldwidewere pre-adapted to this new host. It is nowknown that some variation exists in the abilityof varroa to reproduce on A. cerana, depend-ing on the bee subspecies (Fuchs et al., 2000).Likewise, Warrit et al. (2006) suggest that pop-ulations of varroa on different host popula-tions are genetically differentiated and may beadapted to specific characteristics of their lo-cal host bee populations. Understanding theseadaptive characteristics may eventually lead tonew ways of controlling invasive haplotypeson A. mellifera.
4.5. Implications for world apiculture
New V. destructor haplotypes that colonizeA. mellifera may show slight differences intheir life cycles, which cause them to becomemore virulent than the current K1-1 and J1-1. Indeed, J1-1 is assumed to be far less viru-lent to A. mellifera than K1-1 (de Guzman andRinderer, 1999) and is thought to be displac-ing J1-1 in places where the two are sympatric
(de Guzman et al., 1997; Anderson, 2000;Garrido et al., 2003). These observations cau-tion against the free movement of honeybeesand signal the need for strict and proper quar-antine for the trade of live honeybees betweencountries.
ACKNOWLEDGEMENTS
We thank Prof. Z. Zeng and Prof. Wongsiri forproviding sampling facilities in respectively China,and Thailand and S. Cros-Arteil and C. Moreaufor help in sequencing. A part of the sequencesin this work are from the graduate diploma, Uni-versity Montpellier II, defended by J. Clementin 2004. Funding was provided by the Europeangrant FEOGA (Fonds Europeen d’Orientation etde Garantie Agricole) ID B24000059 to MN andYLC, and a USDA-SCP grant to ZYH. Data used inthis work were (partly) produced through moleculargenetic analysis technical facilities of the IFR119“Montpellier Environnement Biodiversité”.
Nouveaux types asiatiques de Varroa destructor :une menace potentielle pour l’apiculture mon-diale.
Varroa destructor / Apis mellifera / Asie / risquesanitaire / haplotype / ADN mitochondrial
Zusammenfassung – Neue asiatische Typen vonVarroa destructor: eine potentiell neue, weltwei-te Bedrohung für die Bienenhaltung. Die Var-roamilbe, Varroa destructor, ist ein gut adaptier-ter Parasit der Östlichen Honigbiene (Apis cera-na), insbesondere in den nördlichen Regionen desasiatischen Festlands. Mit ihrem Wechsel auf dieWestliche Honigbiene (A. mellifera) im letzen Jahr-hundert kam es zu einer dramatischen Ausbrei-tung dieser Milbe. Als verantwortlich für den Pa-rasitenübergang werden zwei mitochondriale (mt)Haplotypen (K1 und J1) von Varroa angesehen. Be-reits erhobene molekulare Daten deuten darauf hin,dass es sich hierbei, aufgrund der zeitlichen Zu-sammenhänge dieser Wirtswechsel, um zwei par-tiell isolierte Klone handelt. Mittels Genotypisie-rung von V. destructor Isolaten aus Regionen, indenen die Wirtswechsel der J1 und K1 Milben zu-erst stattgefunden hatten, sowie einer weiträumige-ren Untersuchung des natürlichen Ausbreitungsge-biets von V. destructor in Asien, versuchten wir inder vorliegenden Studie ein genaueres Bild überden Befall von A. mellifera durch V. destructorzu erhalten. Jede Milbenprobe wurde zunächst an-hand des publizierten 458 Basenpaarfragments des
192 M. Navajas et al.
mitochondrialen Gens Cytochromoxidase 1 (cox1)genetisch charakterisiert, um neue Varianten bereitsbekannten Haplotypen zuordnen zu können. Umdie genetische Variabilität der Milben besser erfas-sen zu können, wurde im nächsten Schritt ein 2700Nukleotide langes Fragment sequenziert, das viermitochondriale Gene beinhaltet: cox1, Cytochro-moxidase III (cox3), ATP Synthase 6 (atp6) undCytochrom b (cytb). Wir untersuchten Varroamil-ben aus Asien, die sowohl die Östliche (21 Völ-ker) als auch die Westliche Honigbiene (11 Völker)befallen hatten (Tab. I). Milben mit identischer Se-quenz des 458 Basenpaare (bp) langen cox1 Frag-ments wurden als Mitglieder jeweils einer der sie-ben Haplogruppen (K1, J1, V1, C1, C2, C3, L1)betrachtet. Insgesamt fanden wir 18 mt Haplotypen(Varianten der zusammenhängenden Sequenzen in-nerhalb einer Haplogruppe (Tab. III). Von diesenfanden wir 12 auf A. cerana und 6 auf A. mellifera(Tab. I, Abb. 1). Obwohl wir nur eine neue Haplo-gruppe fanden (C3 in Tab. I und III), konnten wir12 neue Haplotypen innerhalb der Haplogruppenerkennen, für die bisher keine genetische Variati-on bekannt war. Dies zeigt, dass Varroamilben inAsien genetisch wesentlich variabler sind, als bis-her angenommen. Unsere Untersuchung zeigt desweiteren, dass die weltweit auf A. mellifera gefun-denden ursprünglichen K1 und J1 Haplotypen vonV. destructor aus zwei unterschiedlichen Milbenpo-pulationen stammen. Diese sind jeweils durch dieK1 und die J1 Haplogruppenzuordnung definiert(Abb. 2), die im nordöstlichen Asien A. cerana be-fallen. Beide Populationen sind genetisch wesent-lich variabler, als bisher angenommen, und die hierneubeschriebenen Haplotypen dieser Populationenhaben A. mellifera in Asien zwar befallen, sich abernoch außerhalb dieser Region verbreitet. Diese neu-en Haplotypen stellen nun neue potentielle Bedro-hungen für A. mellifera außerhalb Asiens dar. Au-ßerdem stellen diese Beobachtungen eine Warnunggegen die freie Verfrachtung von Honigbienen darund zeigen die Notwendigkeit einer strengen undkorrekten Quarantäne für den internationalen Han-del mit lebenden Honigbienen.
Varroa / Apis mellifera / Apis cerana / mitochon-driale DNA / Diversität
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